BEC Transfection

For plasmids transfection containing siRNA against GSK3 isoforms, BECs were grown until 60–70% confluence in F12 medium without serum and antibiotics for 24 h. Then, transfection was performed with Lipofectamine 2000 from Thermo Fisher Scientific (Waltham, MA, USA); for siGSK3α, we added 3 µg of siRNA-GSK3α plasmid (Sigma–Aldrich) and 3 µg of PLKO.1; for siGSK3β, we added 3 µg of pLKO.1-GSK3β-#1 and 3 µg of PLKO.1; for siGSK3αβ, we added 3 µg of siRNA-GSK3α and 3 µg of pLKO.1-GSK3β-#1 plasmid; and for sicontrol, we added 6 µg of PLKO.1. After 24 h, the culture medium was changed to F12 with low serum (1%), and cells were incubated for 5 days. GSK3α and GSK3β gene-silenced cells were screened by Western blotting. For GSK3α, the targeting sequence was 5′-TACATCTGTTCTCGCTACTA-3′ (nucleotides 1535–1554, pLKO.1-GSK3α), and for GSK3β the targeting sequence was 5′-GAAGTCAGCTATACAGACACT-3′ (nucleotides 587–607, pLKO.1-GSK3β). To express the CREB-dominant-negative mutant, cells were cultured at 70–90% confluence in F12 medium without serum or antibiotics for 24 h; then, 350 ng of plasmid pCF-M1-CREB was transfected with Lipofectamine 2000 or 350 ng of pCF empty vector. pCF-M1-CREB plasmid encodes a CREB mutant in which Ser133 was replaced by Ala133. After 12 h, the medium was changed to fresh F12 and low serum, and cells were incubated for an extended period of 48 h. The medium was changed to F12 without serum or antibiotics, and cells were left for at least 4 h before stimulation with S. aureus. Transfection efficiency was monitored by detecting the FLAG tag present on the CREB-mutant construct in Western blot assays. The transfection efficiency of the empty pCF-M1 vector was monitored by the resistance of cells to Geneticin.